1. 1 UV PHOTOFRAGMENTATION SPECTROSCOPY OF MODEL LIGNIN-ALKALI ION COMPLEXES: EXTENDING LIGNOMICS INTO THE SPECTROSCOPIC REGIME JACOB C. DEAN, NICOLE L. BURKE, JOHN R. HOPKINS, JAMES G. REDWINE, a BIDYUT BISWAS, P. V. RAMACHANDRAN, SCOTT A. MCLUCKEY and TIMOTHY S. ZWIER Department of Chemistry, Purdue University, West Lafayette, IN 47906-2084 U.S.A.; a Present address: Kalsec, Kalamazoo, MI 49006 U.S.A.

2. Lignin: Cell Wall Foundation Aromatic biopolymer concentrated in cell wall. “Lignomics” accomplished with tandem MS. Ionization accomplished with negative ESI or alkali metal complexation. Each monolignol is a natural chromophore, providing potential for UV spectroscopy. p-Hydroxyphenyl Guaiacyl Syringyl β -O-4 β - β Vanholme, R, et. al. Plant Physiol. 2010, 153, 895-905 β-5 MonolignolsDilignolsOligolignols Dean,J, et al. Chem. Sci., 2014, 5, 1940-1955Dean,J, et al. J. Chem. Phys, 2013, 139, 144313.

3. Instrument for Cold Ion Spectroscopy Ions are generated via nano-electrospray ionization (nESI) q2 and q3 are set up as linear ion traps The cold trap is a 22-pole ion trap held at ~5K

4. UV photofragment spectroscopy of cryocooled ions (M+Li) +* (M+Li) + S0S0 S1S1 Dissociation threshold Scan UV laser Unimolecular Dissociation (Frag2+Li) + +Y Internal Conversion (M+Li) +‡ (Frag1+Li) + + X Excited State Fragmentation Conformer A Conformer B A B Protonated tyrosine A B

5. IR-induced Fragment Ion Gain Spectroscopy (IRFIG) Unimolecular Dissociation (M+Li) + (M+Li) +* (Frag2+Li) + +Y S0S0 S1S1 Internal Conversion (M+Li) +‡ Dissociation threshold Fix UV dissociation laser to the red of cold transitions Scan IR Laser (Frag1+Li) + + X  t=100 ns Excited State Fragmentation UV wavelength fixed IR produces “warm” ion gain signal

6. (erythro) β-O-4+Li + UV Spectroscopy Three conformer “terraces” at 34985, 35083, and 35161 cm -1 present.

7. (erythro) β-O-4+Li + IR IRFIG spectrum shows only three bands, indicating single dominant linkage conformation for all three conformers. Cation solvated by four oxygen atoms, making binding at this linkage most likely in an oligomer with multiple linkage types. 1 2 3 123

8. β-O-4 Na + UV Two or three conformers present, as with lithiated species, albeit at lower frequencies with more congestion.

9. β-O-4 Na + IR As with lithiated species, single linkage binding conformation similar to B-O-4 Li+ in cold trap. Conformation essentially identical to lithiated species.

10. Pinoresinol + Li + UV and IR (  linkage) First UV transition at 34959 wavenumbers, shifted down by 350 wavenumbers compared with neutral due to metal cation complexation. Second larger transition at 35199 wavenumbers DFT/M05-2X/6-31+G(d) theory for calculations

11. Pinoresinol +Li + UVPD UV Photofragment MS 305 Da peak due to loss of methoxy and OH groups. 214 and 159 Da peaks two separate fragments from same pathway. Relative abundances show higher binding energy of Li cation for the 214 fragment. Parent (365 Da)

12. Pinoresinol + Na + Spectroscopy First sharp transition at 35258 cm -1. Absorption spectrum spans 300 cm -1 to the red indicating possible unresolved bands or excited state processes. Two distinct bands in IRFIG spectrum, compared with only one for lithiated species. Could be due to different conformers or isomers.

13. Pinoresinol+Na + UVPD MS Excited state surfaces steering fragmentation pathways Even slightly different metal ions results in different fragmentation

14. Structural Perturbation by Alkali Metal Complexation Li + Loss of all linkage H-bonds Complete charge solvation by linkage β-O-4 ideal binding site in oligomers β-O-4Li + β-O-4Na + Pino.Li + Pino.Na + Binding E. (eV) 3.692.682.661.88 R O-M (Å)1.972.301.892.26

15. β-O-4 Li + CID vs. UVPD m/z = 279 279 Da peak most prominent fragment in both CID and UVPD of lithiated β-O-4 dilignol. UVPD yields 294 and 312 Da fragments not seen in CID. UVPD CID taken with AB SCIEX QTRAP 4000 Parent (327 Da)

16. β-O-4 Na + CID vs. UVPD m/z =143 UVPD of β-O-4 Na + results in substantially different fragments vs. lithiated species. UVPD fragments different from CID fragments: 143, 156, and 172 Da peaks not seen in CID spectrum. CID UVPD Na+ 343 [M+Na + ] m/z 173

17. Conclusions  Ion spectroscopy adds another dimension to lignomics.  Dilignol transitions resolvable with ion spectroscopy.  Photofragmentation mass spectrometric signatures differ compared to CID.  UVPD of lithiated and sodiated β -O-4 dilignol markedly different. In both cases UVPD provides complementary fragmentation to CID.  Different features found with different linkages.  Future work includes studies of negative ion dilignols.

18. Acknowledgements NSF CRIF CHE-0820766 NSF CHE-1213289 DOE FG02-00ER15105 All group members of the McLuckey and Zwier groups. McLuckey Group Zwier Group